Novel Target for Regulating Multiple Sclerosis

ABSTRACT

Methods are provided for decreasing demyelinating inflammatory disease in a subject by inhibiting the activity of chemokine-like receptor 1 (CMKLR1). Methods are also provided for screening for agents that find use in treating demyelinating inflammatory disease in a subject.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contract AI059635awarded by the National Institutes of Health. The Government has certainrights in this invention.

INTRODUCTION

Human chemokine-like receptor-1 (CMKLR1), a recently de-orphanedG-protein-coupled receptor (GPCR), is specifically expressed on in vitromonocyte-derived dendritic cells, ex vivo macrophages, and circulatingplasmacytoid dendritic cells (pDCs) [Zabel, et al. J Immunol (2005)174(1):244-51; Vermi, et al. J Exp Med (2005) 201(4):509-15]. Thenatural ligand for CMKLR1, chemerin, was recently discovered [Zabel, etal. J Immunol (2005) 174(1):244-51; Wittamer, V., et al., J Exp Med(2003) 198(7): 977-85; Meder, W., et al., FEBS Lett (2003)555(3):495-9.]. Chemerin has been isolated from ascitic fluid (ovariancarcinoma), inflamed synovial fluid, hemofiltrate, and normal serum[Zabel, et al. J Immunol (2005) 174(1):244-51; Wittamer, V., et al., JExp Med (2003) 198(7): 977-85; Meder, W., et al., FEBS Lett (2003)555(3):495-9.]. Chemerin, a heparin binding protein, initially exists inits pro-form, which is 163 amino acids long. Cleavage of pro-chemerin byserine proteases of inflammatory, coagulation, and fibrinolytic cascadesresults in the loss of the last 6-11 C-terminal amino acids. Thisproteolytic cleavage, which can be at a number of different sites inpro-chemerin, generates active chemerin and leads to a potent increasein ligand activity. This results in the increased migration of CMKLR1bearing cells (e.g., macrophages) to chemerin [Wittamer, V., et al., JImmunol (2005) 175(1):487-93, Zabel, B. A., et al., J Biol Chem (2005)280(41): 34661-6]. Chemerin thus acts as a macrophage and dendritic cell(DC) recruiting factor through its interaction with CMKLR1.

While CMKLR1 does not bind to chemokines, it has been reported thatresolvin E1 (RvE1), a bioactive lipid generated upon aspirin-triggeredenzymatic processing of omega-3 fatty acids, is a lipid ligand forCMKLR1 [Hasturk, et al. FASEB J. (2006) 20(2):40′-3; SerhanProstaglandins Leukot Essent Fatty Acids. (2005) 73(3-4):141-62].

Relevant Literature

The use of small molecules to block chemoattractant receptors isreviewed by Baggiolini and Moser (1997) J. Exp. Med. 186:1189-1191.

The sequence of chemerin (retinoic acid receptor responder 2 (RARRES2)II; tazarotene induced gene 2 product (TIG2)) may be found in Genbank,accession number NM_(—)002889. The sequence of CMKLR1 may be found inGenbank, accession number Y14838, and is described by Samson et al.(1998) Eur J. Immunol. 28(5):1689-700. The sequence of a CMKLR1 ligand,mammalian chemerin, may be found in Genbank, accession numberNM_(—)002889.

SUMMARY OF THE INVENTION

The present invention is drawn to methods for decreasing demyelinatinginflammatory disease in a subject by administering agents that decreaseCMKLR1 activity. The therapeutic methods of the invention are useful forthe treatment or prevention of MS and other diseases, e.g. experimentalanimal models such as experimental autoimmune encephalomyelitis (EAE).Inhibitors of CMKLR1 include, but are not limited to, agents thatinterfere with the interaction of CMKLR1 with its natural ligands,agents that reduce CMKLR1 expression (e.g., by reducing transcription orby inducing cell surface receptor desensitization and/orinternalization), agents that reduce expression of endogenous ligands ofCMKLR1, and agents that inhibit intracellular signals initiated by thebinding of CMKLR1 with its ligands. Inhibitors include, withoutlimitation, monoclonal antibodies, small molecules, chimericproteins/peptides, bioactive peptides, and interfering RNA.

The present invention is also drawn to methods of screening for agentsthat can decrease demyelinating inflammatory disease when administeredto a subject. In general, the screening method is designed to determinewhether an agent can antagonize CMKLR1 activity in a cell. In certainembodiments, a cell expressing CMKLR1 (e.g., cells that normally expressCMKLR1 or those that are genetically engineered to express CMKLR1) iscontacted to a candidate agent and its response to a CMKLR1 ligand(s) isevaluated (e.g., chemotaxis, receptor/ligand binding, target geneexpression, signaling responses, etc.). In certain other embodiments, acell expressing CMKLR1 or a ligand is contacted to an agent and theexpression level of CMKLR1 or its ligand is evaluated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Clinical EAE in CMKLR1 KO mice. EAE was induced by activeimmunization and mice were monitored daily for clinical disease. Dataare pooled from five independent experiments and are presented as meanclinical±s.e.m. versus time

FIG. 2. Detection of CMKLR1⁺ dendritic cells and microglia in thecentral nervous system (CNS) of mice with EAE. Mononuclear cells wereisolated from the spinal cords of 3 mice with acute EAE and pooled foranalysis. CD45^(hi)CD3⁻CD19⁻ cells were analyzed for expression ofCD11b, CD11c, and B220. CD11c^(hi)CD11b⁺B220⁻ mDC expressed CMKLR1 whileCD11c^(int)CD11 b⁻B220⁺ pDC were CMKLR1-negative.CD3⁻CD19⁻CD11b⁺CD45^(lo) microglia expressed mCMKLR1. A representativedata set of three independent experiments with similar results is shown.

FIG. 3. anti-mCMKLR1 mAbs block chemerin-mediated chemotaxis in vitro.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

As summarized above, the present invention is drawn to methods fortreating demyelinating inflammatory disease in a subject byadministering an agent that antagonizes the activity of chemokine-likereceptor 1 (CMKLR1) and/or a CMKLR1 ligand (e.g., chemerin or otherendogenous CMKLR1 ligands. As such, the methods of the invention finduse in treating EAE or MS in a subject. Methods of screening for agentsthat regulate demyelinating inflammatory disease are also provided.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

“Activity” of CMKLR1 shall mean any signaling or binding functionperformed by that protein.

“Antibody” shall include, by way of example, both naturally occurringand non-naturally occurring antibodies. Specifically, this term includespolyclonal and monoclonal antibodies, and fragments thereof.Furthermore, this term includes chimeric antibodies and wholly syntheticantibodies, and fragments thereof. Monoclonal antibodies are providedthat bind to CMKLR1 and block its activity. In some embodiments theantibody binds to the epitope that is bound by the monoclonal antibodyBZ186. The hybridoma cell line producing BZ186 may be obtained from theAmerican Type Culture Collection, deposit ______. In other embodimentsthe monoclonal antibody binds to human counterpart of the epitoperecognized by BZ186.

“Anti-sense nucleic acid” shall mean any nucleic acid which, whenintroduced into a cell, specifically hybridizes to at least a portion ofan mRNA in the cell encoding a protein (“target protein”) whoseexpression is to be inhibited, and thereby inhibits the target protein'sexpression.

“Comparable cell” shall mean a cell whose type is identical to that ofanother cell to which it is compared. Examples of comparable cells arecells from the same cell line.

“Expressible nucleic acid” shall mean a nucleic acid encoding a nucleicacid of interest and/or a protein of interest, which nucleic acid is anexpression vector, plasmid or other construct which, when placed in acell, permits the expression of the nucleic acid or protein of interest.Expression vectors and plasmids are well known in the art.

“Inhibiting” the onset of a disorder shall mean either lessening thelikelihood of the disorders onset, or preventing the onset of thedisorder entirely. In the preferred embodiment, inhibiting the onset ofa disorder means preventing its onset entirely. As used herein, onsetmay also refer to deterioration in a patient that haschronic/progressive disease, or relapse in a patient that has ongoingrelapsing-remitting disease.

The methods of the invention may be specifically applied to individualsthat have been diagnosed with an autoimmune disease, e.g. achronic/progressive or relapsing-remitting disease such as MS or EAE.Treatment is aimed at the treatment or prevention of relapses, which arean exacerbation of a pre-existing condition.

“Inhibiting” the expression of a gene in a cell shall mean eitherlessening the degree to which the gene is expressed, or preventing suchexpression entirely.

“Nucleic acid” shall mean any nucleic acid molecule, including, withoutlimitation, DNA, RNA and hybrids thereof. The nucleic acid bases thatform nucleic acid molecules can be the bases A, C, G, T and U, as wellas derivatives thereof. Derivatives of these bases are well known in theart, and are exemplified in PCR Systems, Reagents and Consumables(Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc.,Branchburg, N.J., USA).

Active fragments of CMKLR1 share a functional or binding property withfull length CMKLR1.

Epitopic fragments of CMKLR1 bind to a monoclonal antibody that binds tofull length CMKLR1, including native or denatured forms of the protein.

“Specifically hybridize” to a nucleic acid shall mean, with respect to afirst nucleic acid, that the first nucleic acid hybridizes to a secondnucleic acid with greater affinity than to any other nucleic acid.

“Specifically inhibit” the expression of a protein shall mean to inhibitthat protein's expression or activity (a) more than the expression oractivity of any other protein, or (b) more than the expression oractivity of all but 10 or fewer other proteins.

“Subject” or “patient” shall mean any animal, such as a human, non-humanprimate, mouse, rat, guinea pig or rabbit.

“Suitable conditions” shall have a meaning dependent on the context inwhich this term is used. That is, when used in connection with anantibody, the term shall mean conditions that permit an antibody to bindto its corresponding antigen. When this term is used in connection withnucleic acid hybridization, the term shall mean conditions that permit anucleic acid of at least 15 nucleotides in length to hybridize to anucleic acid having a sequence complementary thereto. When used inconnection with contacting an agent to a cell, this term shall meanconditions that permit an agent capable of doing so to enter a cell andperform its intended function. In one embodiment, the term “suitableconditions” as used herein means physiological conditions.

“Treating” a disorder shall mean slowing, stopping or reversing thedisorders progression. In the preferred embodiment, treating a disordermeans reversing the disorders progression, ideally to the point ofeliminating the disorder itself. As used herein, ameliorating a disorderand treating a disorder are equivalent.

The term “immune” response is the development of a beneficial humoral(antibody mediated) and/or a cellular (mediated by antigen-specific Tcells or their secretion products) response directed against CMKLR1 in arecipient patient. Such a response can be an active response induced byan “immunogen” that is capable of inducing an immunological responseagainst itself on administration to a mammal, optionally in conjunctionwith an adjuvant.

Methods of the Invention

The present invention provides methods for treating autoimmune disease,including inflammatory demyelinating diseases, such multiple sclerosis;etc. These methods comprise administering to the subject having anautoimmune condition, e.g. a demyelinating condition; an effectiveamount of an inhibitor of CMKLR1.

In some embodiments, a method is provided for inhibiting autoimmunediseases in a subject, the method comprising administering to thesubject a prophylactically effective amount of a nucleic acid thatspecifically reduces levels of CMKLR1, e.g. an anti-senseoligonucleotide, siRNA, and the like.

In other embodiments, a method is provided for inhibiting inflammatorydemyelinating disease in a subject, the method comprising administeringto the subject a therapeutically effective amount of an anti-CMKLR1antibody or antigen-binding portion thereof.

In other embodiments, the method comprising administering to saidsubject an agent that downregulates the expression, or inhibits theactivity of, a ligand of CMKLR1, which ligand includes, withoutlimitation, chemerin. In these methods, the CMKLR1-expressing cell canbe, without limitation, a macrophage; a dendritic cell; or a microglialcell.

This invention can utilize a method for reducing the amount of CMKLR1 ina CMKLR1-expressing cell comprising introducing into the cell a nucleicacid which specifically inhibits CMKLR1 expression in the cell. In oneembodiment, this method further reduces the amount of CMKLR1 secreted bya CMKLR1-secreting cell. In this method, the nucleic acid can be, forexample, DNA or RNA. In addition, the nucleic acid can be an anti-sensenucleic acid that hybridizes to CMKLR1-encoding mRNA, an siRNA thatinhibits CMKLR1 expression, or a catalytic nucleic acid that cleavesCMKLR1-encoding mRNA. CMKLR1 expression can also be inhibited using zincfinger proteins or nucleic acids encoding the same as described in WO00100409. Alternatively, inhibition of expression can be achieved usingsiRNAs as described by WO 99132619, Elbashir, EMBO J. 20, 6877-6888(2001) and Nykanen et al., Cell 107, 309-321 (2001); WO 01129058.

The antisense reagent may be antisense oligonucleotides (ODN),particularly synthetic ODN having chemical modifications from nativenucleic acids, or nucleic acid constructs that express such antisensemolecules as RNA. The antisense sequence is complementary to thetargeted miRNA, and inhibits its expression. One or a combination ofantisense molecules may be administered, where a combination maycomprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target miRNA sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 25, usually not morethan about 23-22 nucleotides in length, where the length is governed byefficiency of inhibition, specificity, including absence ofcross-reactivity, and the like.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993) supra. and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature that alter thechemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The alpha.-anomer of deoxyribose may be used, where the baseis inverted with respect to the natural .beta.-anomer. The 2′-OH of theribose sugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars,which provides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

Anti-sense molecules of interest include antagomir RNAs, e.g. asdescribed by Krutzfeldt et al., supra., herein specifically incorporatedby reference. Small interfering double-stranded RNAs (siRNAs) engineeredwith certain ‘drug-like’ properties such as chemical modifications forstability and cholesterol conjugation for delivery have been shown toachieve therapeutic silencing of an endogenous gene in vivo. To developa pharmacological approach for silencing miRNAs in vivo, chemicallymodified, cholesterol-conjugated single-stranded RNA analoguescomplementary to miRNAs were developed, termed ‘antagomirs’. AntagomirRNAs may be synthesized using standard solid phase oligonucleotidesynthesis protocols. The RNAs are conjugated to cholesterol, and mayfurther have a phosphorothioate backbone at one or more positions.

Also of interest in certain embodiments are RNAi agents. Inrepresentative embodiments, the RNAi agent targets the precursormolecule of the microRNA, known as pre-microRNA molecule. By RNAi agentis meant an agent that modulates expression of microRNA by a RNAinterference mechanism. The RNAi agents employed in one embodiment ofthe subject invention are small ribonucleic acid molecules (alsoreferred to herein as interfering ribonucleic acids), i.e.,oligoribonucleotides, that are present in duplex structures, e.g., twodistinct oligoribonucleotides hybridized to each other or a singleribooligonucleotide that assumes a small hairpin formation to produce aduplex structure. By oligoribonucleotide is meant a ribonucleic acidthat does not exceed about 100 nt in length, and typically does notexceed about 75 nt length, where the length in certain embodiments isless than about 70 nt. Where the RNA agent is a duplex structure of twodistinct ribonucleic acids hybridized to each other, e.g., an siRNA, thelength of the duplex structure typically ranges from about 15 to 30 bp,usually from about 15 to 29 bp, where lengths between about 20 and 29bps, e.g., 21 bp, 22 bp, are of particular interest in certainembodiments. Where the RNA agent is a duplex structure of a singleribonucleic acid that is present in a hairpin formation, i.e., a shRNA,the length of the hybridized portion of the hairpin is typically thesame as that provided above for the siRNA type of agent or longer by 4-8nucleotides. The weight of the RNAi agents of this embodiment typicallyranges from about 5,000 daltons to about 35,000 daltons, and in manyembodiments is at least about 10,000 daltons and less than about 27,500daltons, often less than about 25,000 daltons.

dsRNA can be prepared according to any of a number of methods that areknown in the art, including in vitro and in vivo methods, as well as bysynthetic chemistry approaches. Examples of such methods include, butare not limited to, the methods described by Sadher et al. (Biochem.Int. 14:1015, 1987); by Bhattacharyya (Nature 343:484, 1990); and byLivache, et al. (U.S. Pat. No. 5,795,715), each of which is incorporatedherein by reference in its entirety. Single-stranded RNA can also beproduced using a combination of enzymatic and organic synthesis or bytotal organic synthesis. The use of synthetic chemical methods enableone to introduce desired modified nucleotides or nucleotide analogs intothe dsRNA. dsRNA can also be prepared in vivo according to a number ofestablished methods (see, e.g., Sambrook, et al. (1989) MolecularCloning: A Laboratory Manual, 2nd ed.; Transcription and Translation (B.D. Hames, and S. J. Higgins, Eds., 1984); DNA Cloning, volumes I and II(D. N. Glover, Ed., 1985); and Oligonucleotide Synthesis (M. J. Gait,Ed., 1984, each of which is incorporated herein by reference in itsentirety).

In certain embodiments, instead of the RNAi agent being an interferingribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAiagent may encode an interfering ribonucleic acid, e.g., an shRNA, asdescribed above. In other words, the RNAi agent may be a transcriptionaltemplate of the interfering ribonucleic acid. In these embodiments, thetranscriptional template is typically a DNA that encodes the interferingribonucleic acid. The DNA may be present in a vector, where a variety ofdifferent vectors are known in the art, e.g., a plasmid vector, a viralvector, etc.

As indicated above, the antisense agent can be introduced into thetarget cell(s) using any convenient protocol, where the protocol willvary depending on whether the target cells are in vitro or in vivo. Anumber of options can be utilized to deliver the dsRNA into a cell orpopulation of cells such as in a cell culture, tissue, organ or embryo.For instance, RNA can be directly introduced intracellularly. Variousphysical methods are generally utilized in such instances, such asadministration by microinjection (see, e.g., Zernicka-Goetz, et al.(1997) Development 124:1133-1137; and Wianny, et al. (1998) Chromosoma107: 430-439). Other options for cellular delivery includepermeabilizing the cell membrane and electroporation in the presence ofthe dsRNA, liposome-mediated transfection, or transfection usingchemicals such as calcium phosphate. A number of established genetherapy techniques can also be utilized to introduce the dsRNA into acell. By introducing a viral construct within a viral particle, forinstance, one can achieve efficient introduction of an expressionconstruct into the cell and transcription of the RNA encoded by theconstruct.

For example, the inhibitory agent can be fed directly to, injected into,the host organism containing the target gene. The agent may be directlyintroduced into the cell (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, etc. Methods for oral introductioninclude direct mixing of RNA with food of the organism. Physical methodsof introducing nucleic acids include injection directly into the cell orextracellular injection into the organism of an RNA solution. The agentmay be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of the agent may yield more effective inhibition; lowerdoses may also be useful for specific applications.

When liposomes are utilized, substrates that bind to a cell-surfacemembrane protein associated with endocytosis can be attached to theliposome to target the liposome to macrophages or dendritic cells and tofacilitate uptake. Examples of proteins that can be attached includecapsid proteins or fragments thereof that bind to macrophages ordendritic cells, antibodies that specifically bind to cell-surfaceproteins on macrophages or dendritic cells that undergo internalizationin cycling and proteins that target intracellular localizations within Tcells. Gene marking and gene therapy protocols are reviewed by Andersonet al. (1992) Science 256:808-813.

In certain embodiments, a hydrodynamic nucleic acid administrationprotocol is employed. Where the agent is a ribonucleic acid, thehydrodynamic ribonucleic acid administration protocol described indetail below is of particular interest. Where the agent is adeoxyribonucleic acid, the hydrodynamic deoxyribonucleic acidadministration protocols described in Chang et al., J. Virol. (2001)75:3469-3473; Liu et al., Gene Ther. (1999) 6:1258-1266; Wolff et al.,Science (1990) 247: 1465-1468; Zhang et al., Hum. Gene Ther. (1999)10:1735-1737: and Zhang et al., Gene Ther. (1999) 7:1344-1349; are ofinterest.

Additional nucleic acid delivery protocols of interest include, but arenot limited to: those described in U.S. patents of interest include U.S.Pat. Nos. 5,985,847 and 5,922,687 (the disclosures of which are hereinincorporated by reference); WO/11092; Acsadi et al., New Biol. (1991)3:71-81; Hickman et al., Hum. Gen. Ther. (1994) 5:1477-1483; and Wolffet al., Science (1990) 247: 1465-1468; etc.

In another embodiment, relapse of an autoimmune disease in a subject isinhibited or prevented by administering to the subject aprophylactically or therapeutically effective amount of an anti-CMKLR1antibody or antigen-binding portion thereof.

Determining a therapeutically or prophylactically effective amount ofthe CMKLR1 inhibitor compositions can be done based on animal data usingroutine computational methods. In one embodiment, the therapeutically orprophylactically effective amount contains between about 0.1 mg andabout 1 g of nucleic acid or protein, as applicable. In anotherembodiment, the effective amount contains between about 1 mg and about100 mg of nucleic acid or protein, as applicable. In a furtherembodiment, the effective amount contains between about 10 mg and about50 mg of the nucleic acid or protein, as applicable.

In this invention, administering the instant compositions can beeffected or performed using any of the various methods and deliverysystems known to those skilled in the art. The administering can beperformed, for example, intravenously, orally, via implant,transmucosally, transdermally, intramuscularly, intrathecally, andsubcutaneously. The following delivery systems, which employ a number ofroutinely used pharmaceutical carriers, are only representative of themany embodiments envisioned for administering the instant compositions.

Injectable drug delivery systems include solutions, suspensions, gels,microspheres and polymeric injectables, and can comprise excipients suchas solubility-altering agents (e.g., ethanol, propylene glycol andsucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantablesystems include rods and discs, and can contain excipients such as PLGAand polycaprylactone. CMKLR1 or nucleic acids of the invention can alsobe administered attached to particles using a gene gun.

Oral delivery systems include tablets and capsules. These can containexcipients such as binders (e.g., hydroxypropylmethylcellulose,polyvinyl pyrilodone, other cellulosic materials and starch), diluents(e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers andcellulosic materials) and lubricating agents (e.g., stearates and talc).

Transmucosal delivery systems include patches, tablets, suppositories,pessaries, gels and creams, and can contain excipients such assolubilizers and enhancers (e.g., propylene glycol, bile salts and aminoacids), and other vehicles (e.g., polyethylene glycol, fatty acid estersand derivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

Dermal delivery systems include, for example, aqueous and nonaqueousgels, creams, multiple emulsions, microemulsions, liposomes, ointments,aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon basesand powders, and can contain excipients such as solubilizers, permeationenhancers (e.g., fatty acids, fatty acid esters, fatty alcohols andamino acids), and hydrophilic polymers (e.g., polycarbophil andpolyvinylpyrolidone). In one embodiment, the pharmaceutically acceptablecarrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systemsinclude vehicles such as suspending agents (e.g., gums, xanthans,cellulosics and sugars), humectants (e.g., sorbitol), solubilizers(e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g.,sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservativesand Jun. 2, 2005 antioxidants (e.g., parabens, vitamins E and C, andascorbic acid), anti-caking agents, coating agents, and chelating agents(e.g., EDTA).

Conditions for Analysis and Therapy

The compositions and methods of the invention find use in combinationwith a variety of demyelinating autoimmune conditions, includingchronic/progressive and relapsing demyelinating autoimmune diseases.Generally patients for the methods of the present invention arediagnosed as having an autoimmune condition, e.g. a relapsing-remittingautoimmune condition, prior to treatment. The inhibition of CMKLR1decreases the severity or incidence of relapses in such patients.

Multiple sclerosis (MS) is characterized by various symptoms and signsof CNS dysfunction, with remissions and recurring exacerbations. Themost common presenting symptoms are paresthesias in one or moreextremities, in the trunk, or on one side of the face; weakness orclumsiness of a leg or hand; or visual disturbances, e.g. partialblindness and pain in one eye (retrobulbar optic neuritis), dimness ofvision, or scotomas. Other common early symptoms are ocular palsyresulting in double vision (diplopia), transient weakness of one or moreextremities, slight stiffness or unusual fatigability of a limb, minorgait disturbances, difficulty with bladder control, vertigo, and mildemotional disturbances; all indicate scattered CNS involvement and oftenoccur months or years before the disease is recognized. Excess heat mayaccentuate symptoms and signs.

Clinical data alone may be sufficient for a diagnosis of MS. If anindividual has suffered two separate episodes of neurologic symptomscharacteristic of MS, and the individual also has consistentabnormalities on physical examination, a diagnosis of MS can be madewith no further testing. Magnetic resonance imaging (MRI) of the brainand spine is often used during the diagnostic process. MRI shows areasof demyelination (lesions) as bright spots on the image. A substance,called Gadolinium, can be injected into the spinal column to highlightactive plaques and, by elimination, demonstrate the existence ofhistorical lesions not associated with clinical symptoms. This canprovide the evidence of chronic disease needed for a definitivediagnosis of MS. Testing of cerebrospinal fluid (CSF) can provideevidence of chronic inflammation of the central nervous system. The CSFis tested for oligoclonal bands, which are immunoglobulins found in 85%to 95% of people with definite MS. Combined with MRI and clinical data,the presence of oligoclonal bands can help make a definite diagnosis ofMS. Lumbar puncture is the procedure used to collect a sample of CSF.

The brain of a person with MS often responds less actively tostimulation of the optic nerve and sensory nerves. These brain responsescan be examined using visual evoked potentials (VEPs) and somatosensoryevoked potentials (SEPs). Decreased activity on either test can revealdemyelination which may be otherwise asymptomatic. Along with otherdata, these exams can help find the widespread nerve involvementrequired for a definite diagnosis of MS.

In 1996 the United States National Multiple Sclerosis Societystandardized the following four subtype definitions (see Lublin andReingold (1996) Neurology 46(4):907-11, herein specifically incorporatedby reference) as relapsing-remitting; secondary progressive; primaryprogressive; progressive relapsing. The methods of the invention findparticular use in the treatment of ongoing disease, and particularly intreating relapsing forms.

Relapsing-remitting describes the initial course of 85% to 90% ofindividuals with MS. This subtype is characterized by unpredictableattacks (relapses) followed by periods of months to years of relativequiet (remission) with no new signs of disease activity. Deficitssuffered during the attacks may either resolve or may be permanent. Whendeficits always resolve between attacks, this is referred to as “benign”MS.

Secondary progressive describes around 80% of those with initialrelapsing-remitting MS, who then begin to have neurologic declinebetween their acute attacks without any definite periods of remission.This decline may include new neurologic symptoms, worsening cognitivefunction, or other deficits. Secondary progressive is the most commontype of MS and causes the greatest amount of disability.

Primary progressive describes the approximately 10% of individuals whonever have remission after their initial MS symptoms. Decline occurscontinuously without clear attacks. The primary progressive subtypetends to affect people who are older at disease onset.

Progressive relapsing describes those individuals who, from the onset oftheir MS, have a steady neurologic decline but also suffer superimposedattacks; and is the least common of all subtypes.

Treatments for MS include interferon β (Avonex, Betaseron, Rebif),Copaxone (Glatiramer acetate), and anti-VLA4 (Tysabri, natalizumab),which reduce relapse rate and to date have only exhibited a modestimpact on disease progression. MS is also treated with immunosuppressiveagents including methylprednisolone, other steroids, methotrexate,cladribine and cyclophosphamide. Many biological agents, such asanti-IFNgamma antibody, CTLA4-Ig (Abetacept), anti-CD20 (Rituxan), andother anti-cytokine agents are in clinical development for MS.

Peripheral neuropathies may also have a relapsing remitting course, andmay include Miller Fisher syndrome; chronic inflammatory demyelinatingpolyneuropathy (CIDP) with its subtypes classical CIDP, CIDP withdiabetes, CIDP/monoclonal gammopathy of undetermined significance(MGUS), sensory CIDP, multifocal motor neuropathy (MMN), multifocalacquired demyelinating sensory and motor neuropathy or Lewis-Sumnersyndrome, multifocal acquired sensory and motor neuropathy, and distalacquired demyelinating sensory neuropathy; IgM monoclonal gammopathieswith its subtypes Waldenstrom's macroglobulinemia, myelin-associatedglycoprotein-associated gammopathy, polyneuropathy, organomegaly,endocrinopathy, M-protein, skin changes syndrome, mixedcryoglobulinemia, gait ataxia, late-onset polyneuropathy syndrome, andMGUS.

An inhibitory agent may inhibit the activity of CMKLR1 by a variety ofdifferent mechanisms. In certain embodiments, the inhibitory agent isone that binds to the protein CMKLR1 and, in doing so, inhibits itsactivity. In other embodiments, the inhibitory agent prevents expressionor secretion of CMKLR1.

Representative CMKLR1 inhibitory agents include, but are not limited to:antisense oligonucleotides; antibodies; and the like. Other agents ofinterest include, but are not limited to: naturally occurring orsynthetic small molecule compounds of interest, which include numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 50 and less than about 2,500 daltons. Candidate agents comprisefunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Such molecules may beidentified, among other ways, by employing appropriate screeningprotocols.

The inhibitory agent may act on CMKLR1 mRNA to inhibit the activity ofthe target CMKLR1 by reducing the amount of CMKLR1RNA present in thetargeted cells, where the target cell may be present in vitro or invivo. By “reducing the amount of” is meant that the level or quantity ofthe target CMKLR1 in the target cell is reduced by at least about2-fold, usually by at least about 5-fold, e.g., 10-fold, 15-fold,20-fold, 50-fold, 100-fold or more, as compared to a control, i.e., anidentical target cell not treated according to the subject methods.

In another embodiment, the CMKLR1 inhibitor is an antibody. The term“antibody” or “antibody moiety” is intended to include any polypeptidechain-containing molecular structure with a specific shape that fits toand recognizes an epitope, where one or more non-covalent bindinginteractions stabilize the complex between the molecular structure andthe epitope. The term includes monoclonal antibodies, multispecificantibodies (antibodies that include more than one domain specificity),human antibody, humanized antibody, and antibody fragments with thedesired biological activity.

Polyclonal antibodies can be raised by a standard protocol by injectinga production animal with an antigenic composition, formulated asdescribed above. (See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988.) In one such technique, aClass II target antigen comprising an antigenic portion of thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). When utilizing an entireprotein, or a larger section of the protein, antibodies may be raised byimmunizing the production animal with the protein and a suitableadjuvant (e.g., incomplete Freund's, complete Freund's, oil-in-wateremulsions, etc.) Alternatively, for monoclonal antibodies, hybridomasmay be formed by isolating the stimulated immune cells, such as thosefrom the spleen of the inoculated animal. These cells are then fused toimmortalized cells, such as myeloma cells or transformed cells, whichare capable of replicating indefinitely in cell culture, therebyproducing an immortal, immunoglobulin-secreting cell line.

In addition, the antibodies or antigen binding fragments may be producedby genetic engineering. In this technique, as with the standardhybridoma procedure, antibody-producing cells are sensitized to thedesired antigen or immunogen. The messenger RNA isolated from the immunespleen cells or hybridomas is used as a template to make cDNA using PCRamplification. A library of vectors, each containing one heavy chaingene and one light chain gene retaining the initial antigen specificity,is produced by insertion of appropriate sections of the amplifiedimmunoglobulin cDNA into the expression vectors. A combinatorial libraryis constructed by combining the heavy chain gene library with the lightchain gene library. This results in a library of clones, whichco-express a heavy and light chain (resembling the Fab fragment orantigen binding fragment of an antibody molecule). The vectors thatcarry these genes are co-transfected into a host (e.g. bacteria, insectcells, mammalian cells, or other suitable protein production host cell).When antibody gene synthesis is induced in the transfected host, theheavy and light chain proteins self-assemble to produce activeantibodies that can be detected by screening with the antigen orimmunogen.

Antibodies with a reduced propensity to induce a violent or detrimentalimmune response in humans (such as anaphylactic shock), and which alsoexhibit a reduced propensity for priming an immune response which wouldprevent repeated dosage with the antibody therapeutic are preferred foruse in the invention. Thus, humanized, single chain, chimeric, or humanantibodies, which produce less of an immune response when administeredto humans, are preferred for use in the present invention. Also includedin the invention are multi-domain antibodies.

A chimeric antibody is a molecule in which different portions arederived from different animal species, for example those having avariable region derived from a murine mAb and a human immunoglobulinconstant region. Techniques for the development of chimeric antibodiesare described in the literature. See, for example, Morrison et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al. (1984)Nature 312:604-608; Takeda et al. (1985) Nature 314:452-454. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide. See, for example, Huston et al., Science242:423-426; Proc. Natl. Acad. Sci. 85:5879-5883; and Ward et al. Nature341:544-546.

Antibody fragments that recognize specific epitopes may be generated bytechniques well known in the field. These fragments include, withoutlimitation, F(ab′)₂ fragments, which can be produced by pepsin digestionof the antibody molecule, and Fab fragments, which can be generated byreducing the disulfide bridges of the F(ab′)₂ fragments.

Alternatively, single chain antibodies (Fv, as described below) can beproduced from phage libraries containing human variable regions. SeeU.S. Pat. No. 6,174,708. Intrathecal administration of single-chainimmunotoxin, LMB-7 [B3(Fv)-PE38], has been shown to cure ofcarcinomatous meningitis in a rat model. Proc Natl. Acad. Sci. USA 92,2765-9, all of which are incorporated by reference fully herein.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by ficin, pepsin, papain, or otherprotease cleavage. “Fragment,” or minimal immunoglobulins may bedesigned utilizing recombinant immunoglobulin techniques. For instance“Fv” immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

Candidate antibodies can be tested for by any suitable standard means,e.g. ELISA assays, etc. As a first screen, the antibodies may be testedfor binding against the immunogen. After selective binding isestablished, the candidate antibody may be tested for appropriateactivity in an in vivo model. In a preferred embodiment, antibodycompounds may be screened using a variety of methods in vitro and invivo. These methods include, but are not limited to, methods thatmeasure binding affinity to a target, biodistribution of the compoundwithin an animal or cell, or compound mediated cytotoxicity. These andother screening methods known in the art provide information on theability of a compound to bind to, modulate, or otherwise interact withthe specified target and are a measure of the compound's efficacy.

Anti-CMKLR1 antibodies may be administered daily, semi-weekly, weekly,semi-monthly, monthly, etc., at a dose of from about 0.01 mg, from about0.1 mg, from about 1 mg, from about 5 mg, from about 10 mg, from about100 mg or more per kilogram of body weight when administeredsystemically. Smaller doses may be utilized in localized administration,e.g. in direct administration to ocular nerves, etc. Humanized, chimerichuman, or human antibodies are preferred for administering to humanpatients.

Methods of Screening for CMKLR1 Antagonists

Agents that can regulate demyelinating inflammatory disease in a subjectcan be identified by detecting the ability of an agent to antagonize theactivity of CMKLR1. Antagonizing agents include, but are not limited to,agents that interfere with the interaction of CMKLR1 with its naturalligands, agents that reduce CMKLR1 expression (e.g., by reducingtranscription or by inducing cell surface receptor desensitization,internalization and/or degradation), agents that reduce expression ofendogenous ligands of CMKLR1, and agents that inhibit intracellularsignals initiated by the binding of CMKLR1 with its ligands.

In certain embodiments, agents that can reduce demyelinatinginflammatory disease in a subject can be identified by detecting theability of an agent to interfere with (e.g., block) the interaction ofCMKLR1 with its cognate ligand (e.g., chemerin). For example, ascreening assay may be used that evaluates the ability of an agent tobind specifically to CMKLR1 (or its ligand) and prevent receptor:ligandinteraction. Assays to determine affinity and specificity of binding areknown in the art, including competitive and non-competitive assays.Assays of interest include ELISA, RIA, flow cytometry, etc. Bindingassays may use purified or semi-purified protein, or alternatively mayuse primary cells or immortalized cell lines that express CMKLR1. Incertain of these embodiments, the cells are transfected with anexpression construct for CMKLR1. As an example of a binding assay,CMKLR1 is inserted into a membrane, e.g. whole cells, or membranescoating a substrate, e.g. microtiter plate, magnetic beads, etc. Thecandidate agent and soluble, labeled ligand (e.g., chemerin) are addedto the cells, and the unbound components are then washed off. Theability of the agent to compete with the labeled ligand for receptorbinding is determined by quantitation of bound, labeled ligand.Confirmation that the blocking agent does not cross-react with otherchemoattractant receptors may be performed with a similar assay.

CMKLR1 protein sequences are used in screening of candidate compounds(including antibodies, peptides, lipids, small organic molecules, etc.)for the ability to bind to and modulate CMKLR1 activity. Agents thatinhibit or reduce CMKLR1 activity are of interest as therapeutic agentsfor decreasing demyelinating inflammatory disease in a subject whereasagents that activate CMKLR1 activity are of interest as therapeuticagents for increasing demyelinating inflammatory disease in a subject.Such compound screening may be performed using an in vitro model, agenetically altered cell or animal, or purified protein corresponding tochemerin-like chemoattractant polypeptides or a fragment(s) thereof. Onecan identify ligands or substrates that bind to and modulate the actionof the encoded polypeptide.

Polypeptides useful in screening include those encoded by the CMKLR1gene, as well as nucleic acids that, by virtue of the degeneracy of thegenetic code, are not identical in sequence to the disclosed nucleicacids, and variants thereof.

CMKLR1 ligands (e.g., chemerin or resolvin) are used in screening ofcandidate compounds (including antibodies, peptides, lipids, smallorganic molecules, etc.) for the ability to bind to and modulate theligands ability to activate CMKLR1. Agents that inhibit or reduce theability of a CMKLR1 ligand to activate CMKLR1 are of interest astherapeutic agents for decreasing demyelinating inflammatory disease ina subject whereas agents that increase the ability of a CMKLR1 ligand toactivate CMKLR1 activity are of interest as therapeutic agents forincreasing demyelinating inflammatory disease in a subject. Suchcompound screening may be performed using an in vitro model, agenetically altered cell or animal, or purified protein corresponding tochemerin-like chemoattractant polypeptides or a fragment(s) thereof. Onecan identify ligands or substrates that bind to and modulate the actionof the encoded polypeptide.

Polypeptides useful in screening include those encoded by a CMKLR1ligand gene (e.g., chemerin), as well as nucleic acids that, by virtueof the degeneracy of the genetic code, are not identical in sequence tothe disclosed nucleic acids, and variants thereof.

Transgenic animals or cells derived therefrom are also used in compoundscreening. Transgenic animals may be made through homologousrecombination, where the normal locus corresponding to chemerin-likechemoattractant is altered. Alternatively, a nucleic acid construct israndomly integrated into the genome. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, yeastartificial chromosomes (YACs), and the like. A series of small deletionsand/or substitutions may be made in the coding sequence to determine therole of different exons in receptor binding, signal transduction, etc.Specific constructs of interest include antisense sequences that blockexpression of the targeted gene and expression of dominant negativemutations. A detectable marker, such as lac Z or GFP, may be introducedinto the locus of interest, where up-regulation of expression willresult in an easily detected change in phenotype. One may also providefor expression of the target gene or variants thereof in cells ortissues where it is not normally expressed or at abnormal times ofdevelopment, for example by overexpressing in neural cells. By providingexpression of the target protein in cells in which it is not normallyproduced, one can induce changes in cell behavior.

Compound screening identifies agents that modulate CMKLR1 activity orfunction. Of particular interest are screening assays for agents thathave a low toxicity for normal human cells. A wide variety of assays maybe used for this purpose, including labeled in vitro protein-proteinbinding assays, electrophoretic mobility shift assays, immunoassays forprotein binding, and the like. Screening for the activity of G-proteincoupled receptors (or GPCRs, of which CMKLR1 is a member) is well knownin the art, and includes assays for measuring any of a number ofdetectible steps, including but not limited to: stimulation of GDP forGTP exchange on a G protein; alteration of adenylate cyclase activity;protein kinase C modulation; phosphatidylinositol breakdown (generatingsecond messengers diacylglycerol, and inositol triphosphate);intracellular calcium flux; activation of MAP kinases; modulation oftyrosine kinases; modulation of gene or reporter gene activity, integrinactivation, or chemotaxis inhibition. A detectable step in a signalingcascade is considered modulated if the measurable activity is altered by10% or more above or below a baseline or control level. The baseline orcontrol level can be the activity in the substantial absence of anactivator (e.g., a ligand) or the activity in the presence of a knownamount of an activator. The measurable activity can be measureddirectly, as in, for example, measurement of cAMP or diacylglycerollevels. Alternatively, the measurable activity can be measuredindirectly, as in, for example, a reporter gene assay. Knowledge of the3-dimensional structure of the encoded protein (e.g., CMKLR1 or aligand, e.g. chemerin), derived from crystallization of purifiedrecombinant protein, could lead to the rational design of small drugsthat specifically inhibit activity. These drugs may be directed atspecific domains and sites.

The term “agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of modulating the physiologicalfunction of CMKLR1 or its ligand. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typicallyone of these concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, lipids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Test agents can be obtained from libraries, such asnatural product libraries or combinatorial libraries, for example.

Libraries of candidate compounds can also be prepared by rationaldesign. (See generally, Cho et al., Pac. Symp. Biocompat. 305-16, 1998);Sun et al., J. Comput. Aided Mol. Des. 12:597-604, 1998); eachincorporated herein by reference in their entirety). For example,libraries of phosphatase inhibitors can be prepared by syntheses ofcombinatorial chemical libraries (see generally DeWitt et al., Proc.Nat. Acad. Sci. USA 90:6909-13, 1993; International Patent PublicationWO 94/08051; Baum, Chem. & Eng. News, 72:20-25, 1994; Burbaum et al.,Proc. Nat. Acad. Sci. USA 92:6027-31, 1995; Baldwin et al., J. Am. Chem.Soc. 117:5588-89, 1995; Nestler et al., J. Org. Chem. 59:4723-24, 1994;Borehardt et al., J. Am. Chem. Soc. 116:373-74, 1994; Ohlmeyer et al.,Proc. Nat. Acad. Sci. USA 90:10922-26, all of which are incorporated byreference herein in their entirety.)

A “combinatorial library” is a collection of compounds in which thecompounds comprising the collection are composed of one or more types ofsubunits. Methods of making combinatorial libraries are known in theart, and include the following: U.S. Pat. Nos. 5,958,792; 5,807,683;6,004,617; 6,077,954; which are incorporated by reference herein. Thesubunits can be selected from natural or unnatural moieties. Thecompounds of the combinatorial library differ in one or more ways withrespect to the number, order, type or types of modifications made to oneor more of the subunits comprising the compounds. Alternatively, acombinatorial library may refer to a collection of “core molecules”which vary as to the number, type or position of R groups they containand/or the identity of molecules composing the core molecule. Thecollection of compounds is generated in a systematic way. Any method ofsystematically generating a collection of compounds differing from eachother in one or more of the ways set forth above is a combinatoriallibrary.

A combinatorial library can be synthesized on a solid support from oneor more solid phase-bound resin starting materials. The library cancontain five (5) or more, preferably ten (10) or more, organic moleculesthat are different from each other. Each of the different molecules ispresent in a detectable amount. The actual amounts of each differentmolecule needed so that its presence can be determined can vary due tothe actual procedures used and can change as the technologies forisolation, detection and analysis advance. When the molecules arepresent in substantially equal molar amounts, an amount of 100 picomolesor more can be detected. Preferred libraries comprise substantiallyequal molar amounts of each desired reaction product and do not includerelatively large or small amounts of any given molecules so that thepresence of such molecules dominates or is completely suppressed in anyassay.

Combinatorial libraries are generally prepared by derivatizing astarting compound onto a solid-phase support (such as a bead). Ingeneral, the solid support has a commercially available resin attached,such as a Rink or Merrifield Resin. After attachment of the startingcompound, substituents are attached to the starting compound.Substituents are added to the starting compound, and can be varied byproviding a mixture of reactants comprising the substituents. Examplesof suitable substituents include, but are not limited to, hydrocarbonsubstituents, e.g. aliphatic, alicyclic substituents, aromatic,aliphatic and alicyclic-substituted aromatic nuclei, and the like, aswell as cyclic substituents; substituted hydrocarbon substituents, thatis, those substituents containing nonhydrocarbon radicals which do notalter the predominantly hydrocarbon substituent (e.g., halo (especiallychloro and fluoro), alkoxy, mercapto, alkylmercapto, nitro, nitroso,sulfoxy, and the like); and hetero substituents, that is, substituentswhich, while having predominantly hydrocarbyl character, contain otherthan carbon atoms. Suitable heteroatoms include, for example, sulfur,oxygen, nitrogen, and such substituents as pyridyl, furanyl, thiophenyl,imidazolyl, and the like. Heteroatoms, and typically no more than one,can be present for each carbon atom in the hydrocarbon-basedsubstituents. Alternatively, there can be no such radicals orheteroatoms in the hydrocarbon-based substituent and, therefore, thesubstituent can be purely hydrocarbon.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin, etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Thecomponents are added in any order that provides for the requisitebinding. Incubations are performed at any suitable temperature,typically between 4 and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening. Typically between 0.1 and 3 hours will besufficient.

Preliminary screens can be conducted by screening for compounds capableof binding to CMKLR1 or its ligand; compounds so identified are possiblemodulators. Compounds capable of binding to CMKLR1 are inhibitors ifthey do not activate the receptor and activators if they do. The bindingassays usually involve contacting CMKLR1 or its ligand with one or moretest compounds and allowing sufficient time for the protein and testcompounds to form a binding complex. Any binding complexes formed can bedetected using any of a number of established analytical techniques.Protein binding assays include, but are not limited to, methods thatmeasure co-precipitation, co-migration on non-denaturingSDS-polyacrylamide gels, and co-migration on Western blots (see, e.g.,Bennet, J. P. and Yamamura, H. I. (1985) “Neurotransmitter, Hormone orDrug Receptor Binding Methods,” in Neurotransmitter Receptor Binding(Yamamura, H. I., et al., eds.), pp. 61-89.

Certain screening methods involve screening for a compound thatmodulates the expression of CMKLR1 or its ligand. Such methods generallyinvolve conducting cell-based assays in which test compounds arecontacted with one or more cells endogenously expressing CMKLR1 or itsligand and then detecting a modulation in expression (e.g., at the mRNAand/or protein level). In certain screening methods, a target cell has areporter gene (e.g., GFP) under the control of the CMKLR1 promoter (orpromoter of its ligand). The level of expression can be compared to abaseline value. The baseline value can be a value for a control sampleor a statistical value that is representative of expression levels for acontrol population. Expression levels can also be determined for cellsthat do not express the CMKLR1 or its ligand, as a negative control.Such cells generally are otherwise substantially genetically the same asthe test cells. Various controls can be conducted to ensure that anobserved activity is authentic including running parallel reactions withcells that lack the reporter construct or by not contacting a cellharboring the reporter construct with test compound.

Certain screening methods involve screening for a compound thatmodulates gene expression normally regulated by CMKLR1 signaling. Incertain embodiments, a cell-based assay is conducted in which a cellexpressing CMKLR1 is contacted to a candidate agent (e.g., a CMKLR1binding agent) and monitored for changes in gene expression that aresimilar, or substantially similar, to those induced by a natural ligandfor CMKLR1. In certain other embodiments, a cell-based assay isconducted in which a cell expressing CMKLR1 is contacted to its naturalligand and a candidate agent and monitored for perturbations in geneexpression. By “perturbations in gene expression”, it is meant that thegene expression changes induced by a CMKLR1 ligand binding to CMKLR1 isaltered when the candidate agent is present.

Certain screening methods involve screening for a compound thatmodulates CMKLR1 signaling events when contacted to a cell expressingCMKLR1. These assays can be carried out in the presence or absence of anatural ligand for CMKLR1. Such methods generally involve monitoring formodulation of downstream signaling events as described above, e.g.,protein phosphorylation, GDP/GTP exchange, etc.

Compounds can also be further validated as described below.

Compounds that are initially identified by any of the foregoingscreening methods can be further tested to validate their apparentactivity. The basic format of such methods involves administering a leadcompound identified during an initial screen to an animal that serves asa model for humans. The animal models utilized in validation studiesgenerally are mammals. Specific examples of suitable animals include,but are not limited to, primates, mice, and rats.

Active test agents identified by the screening methods described hereinthat modulate CMKLR1 activity can serve as lead compounds for thesynthesis of analog compounds. Typically, the analog compounds aresynthesized to have an electronic configuration and a molecularconformation similar to that of the lead compound. Identification ofanalog compounds can be performed through use of techniques such asself-consistent field (SCF) analysis, configuration interaction (CI)analysis, and normal mode dynamics analysis. Computer programs forimplementing these techniques are available. See, e.g., Rein et al.,(1989) Computer-Assisted Modeling of Receptor-Ligand Interactions (AlanLiss, New York).

A functional assay that detects leukocyte chemotaxis may be used forconfirmation. For example, a population of cells that demonstratechemerin chemotaxis (e.g., dendritic cells or monocyte/macrophages) maybe stimulated with chemerin and/or the candidate modulating agent. Anagent that antagonizes CMKLR1 activity will cause a decrease in thelocomotion of the cells in response to chemerin. An agent thatpotentiates CMKLR1 activity will act as a chemotaxis factor in theabsence of chemerin and/or increase the chemotactic response induced bychemerin. Chemotaxis assays of that find use in these methods are knownin the art, examples of which are described in U.S. patent applicationSer. No. 10/958,527, entitled “Family of Cystatin-RelatedChemoattractant Proteins” (incorporated herein by reference in itsentirety). An agent that is a chemoattractant inhibitor will decreasethe concentration of cells at a target site of higher concentration ofchemerin.

EXPERIMENTAL Example 1

The pathology of multiple sclerosis (MS) involves leukocyteextravasation of the blood-brain barrier and associated myelin damage,which leads to impaired nerve function and paralysis. Chemokines,adhesion molecules, and their receptors have been implicated inrecruitment of inflammatory cells to the central nervous system (CNS)during MS. However, the mechanisms that regulate migration of variousleukocyte subsets to the CNS remain poorly understood. Chemokine-likereceptor (CMKLR)-1 (also known as ChemR23 or Dez) is a recentlyde-orphaned chemoattractant receptor that has emerging roles inmacrophage migration during inflammatory processes. In this study, weexamined the role of CMKLR1 in experimental autoimmune encephalomyelitis(EAE), a mouse model of human MS. We found that mice deficient in CMKLR1are resistant to progressive EAE. CMKLR1-deficient mice had reducedinflammatory infiltrates in the brain and spinal cord, with especiallymarked differences in the CNS parenchyma. Together, the data demonstratethat CMKLR1 regulates autoimmune demyelinating disease; and CMKLR1provides a target for blocking development of progressive EAE/MS.

Example 2

Mouse monoclonal antibody BZ186 specifically recognizes mouse serpentineprotein CMKLR1. CMKLR1 possesses high homology with members of thechemoattractant receptor family, and binds the chemoattractant chemerin.CMKLR1 is selectively expressed in macrophages, natural killer (NK)cells, subsets of dendritic cells (DC), and adipocytes. Monoclonalantibodies directed against chemokine receptors are used to determineleukocyte expression profile of receptors during homeostasis orinflammation; role of various receptors in coordinating the immuneresponse; role of various receptors in leukocyte development; identityof other proteins interacting with the chemokine receptor.

This mouse anti-mCMKLR1 antibody can be used in flow cytometry, as ablocking reagent in vitro and in vivo; and in immunocytochemistry andimmunofluorescence. Monoclonal antibody BZ186 (isotype mIgG₁κ) wasgenerated by immunizing a CMKLR1 KO mouse with wild-type totalperitoneal exudate cells. mCMKLR1 is highly expressed on mousemacrophages, which make up ˜30% of total peritoneal exudate cells.Splenocytes and draining lymph node cells were isolated from theimmunized mouse and fused with SP2/0 myeloma cells. Hybridomasupernatants were screened for binding to the mCMKLR1/L1.2 cell line.Hybridomas secreting monoclonal antibodies specific for mCMKLR1 wereisolated and cloned by limiting dilution. Anti-mCMKLR1 mAb BZ186 waspurified from a large batch preparation of hybridoma supernatant. Theantibody is composed of the constant region from mouse immunoglobulinheavy chain isotype G₁ and the constant region from the kappa lightchain. The complementarity-determining regions (CDR) recognize mCMKLR1.mAb BZ186 was shown to stain peritoneal mouse macrophages by flowcytometry, and block mCMKLR1/L1.2 transfectant chemotaxis to chemerin inin vitro transwell migration assays. A modification is made by directlylabeling the antibodies with a fluorophore for use in flow cytometry,eliminating the need for a second-stage reagent. The antibody can alsobe biotinylated, which allows for higher sensitivity in various assays.The mAb can be conjugated to magnetic microbeads, which can be used toseparate and enrich/purify mCMKLR1+ cells.

Importantly, the BZ186 mAb blocks mCMKLR1 functional responses tochemerin.

In a variation of this method, a mouse is similarly immunized with humanmacrophages, and screened for specific binding to the human protein,preferably as presented on the cell surface, wherein a monoclonalantibody is obtained that blocks functional responses to human chemerin.

Example 3

Using the newly developed anti-mCMKLR1 mAb BZ186, we analyzed CMKLR1expression on mouse spinal cord mononuclear cells by flow cytometry.Plasmacytoid dendritic cells, pDC, defined asCD45^(hi)CD3⁻CD19⁻CD11b⁻CD11c^(int)B220⁺, are CMKLR1-negative, whereasmyeloid dendritic cells mDC, defined asCD45^(hi)CD3⁻CD19⁻CD11b⁺CD11c^(hi)B220⁻ and microglia, defined asCD3⁻CD19⁻CD11b⁺CD45^(lo), isolated from the spinal cords of EAE mice areCMKLR1-positive.

Example 4

EAE is examined in CMKLR1 null mice immunized with MOG 35-55 in CFA.These mice are resistant to development of EAE relative to thewild-type. EAE is driven by pathogenic immune responses against myelinproteins and lipids. CMKLR1 may have an inflammatory role.

It is then determined whether treatment with agents that inhibit CMKLR1,including mAb BZ186 or RNAi specific for CMKLR1 or chemerin. To testthis, WT mice with EAE are treated every two days with 10-100 μg of mAbBZ186 administered intravenously or intraperitoneally.

Methods

Mice. CMKLR1 null mice were developed by Deltagen. These null mice weregenerated from ES cells derived from the 129 mouse strain andbackcrossed to the C57BL/6 background. The mice are viable and fertile,with no obvious prenatal defects.

EAE induction. EAE is induced in 8-12 week old female null and WTanimals via subcutaneous immunization with 100 μg myelin oligodendrocyteglycoprotein) peptide, amino acids 35-55 (MOG 35-55) in an emulsionmixed (volume ratio 1:1) with Complete Freund's Adjuvant (containing 4mg/ml of heat-killed Mycobacterium tuberculosis H37Ra). Mice are alsoinjected intravenously with 250-400 ng of Bordetella pertussis toxin(BPT) in PBS at the time of, and two days following immunization. MOG35-55 peptide is synthesized by the Stanford Protein and Nucleic AcidFacility and purified by high performance liquid chromatography (HPLC).Mice (n=8-10 per group) were examined daily for clinical signs of EAEand were scored as followed: 0=no clinical disease, 1=limp tail,2=hindlimb weakness, 3=complete hindlimb paralysis, 4=hindlimb paralysisplus some forelimb paralysis, and 5=moribund or dead.

Histopathology. Brains and spinal cords are dissected from mice, fixedin 10% formalin in PBS and embedded in paraffin. Seven micron thicksections are stained with haematoxylin and eosin to detect inflammatoryinfiltrates and luxol fast blue for demyelination. Inflammatory lesionsin brain, thoracic and lumbar spinal cord sections are counted by anexaminer masked to the treatment status of the animal.

Treatment. WT mice are induced with EAE using MOG 35-55 and pertussistoxin. When mice have hindlimb weakness or paralysis, animals aredivided into two groups balanced for mean clinical disease scores, andthen injected intravenously or intraperitoneally every second day withsaline, pH 7.0, or 10-100 μg monoclonal antibody diluted in saline.

Example 5

EAE is induced in animals as described above. In place of treatment withantibody, the animals are injected with cholesterol conjugated siRNAhaving the sequenceFw:CACCGGAAGATAACCTGCTTCAACACGAATGTTGAAGCAGGTTATCTTCC (SEQ ID NO. 1)Fw:AAAAGGAAGATAACCTGCTTCAACATTCGTGTTGAAGCAGGTTATCTTCC (SEQ ID NO. 2) orwith an adenoviral siRNA construct, and the results are scored asdescribed above.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

What is claimed is:
 1. A method of decreasing demyelinating inflammatory disease in a subject, the method comprising: administering to said subject an effective amount of a chemokine-like receptor 1 (CMKLR1) antagonist.
 2. The method of claim 1, wherein said CMKLR1 antagonist binds to CMKLR1.
 3. The method of claim 2, wherein said CMKLR1 antagonist comprises a natural or altered domain derived from a natural ligand of CMKLR1.
 4. The method of claim 1, wherein said CMKLR1 antagonist is a polypeptide.
 5. The method of claim 4, wherein said polypeptide is an antibody or antigen binding fragment thereof.
 6. The method of claim 1, wherein said CMKLR1 modulatory agent is an siRNA.
 7. The method of claim 1, wherein said antagonizing agent inhibits ligand-induced signaling from CMKLR1 in a cell.
 8. The method of claim 1, wherein said antagonizing agent reduces the expression of CMKLR1 in a cell.
 9. The method of claim 1, wherein said antagonizing agent inhibits transcription of CMKLR1.
 10. A method of screening for an agent that decreases demyelinating inflammatory disease in a subject, said method comprising: contacting said agent to a cell expressing CMKLR1; and evaluating whether said agent antagonizes CMKLR1 activity.
 11. The method of claim 10, wherein said CMKLR1 activity is selected from the group consisting of: chemotaxis, activation of a signaling pathway component, activation of gene or reporter gene expression, and expression of CMKLR1.
 12. The method of claim 10, wherein said method is a high throughput screening method.
 13. The method of claim 10, wherein said cell naturally expresses CMKLR1 or is genetically engineered to express CMKLR1.
 14. The method of claim 10, further comprising validating said agent as a demyelinating inflammatory disease regulator in an animal model. 